Modern silviculture often requires the planting of large numbers of genetically identical plants that have been selected to have advantageous properties. Production of new plants by sexual reproduction, which yields botanic seeds, is usually not feasible. Asexual propagation, via the culturing of somatic or zygotic embryos, has been shown for some species to yield large numbers of genetically identical embryos, each having the capacity to develop into a normal plant.
Somatic cloning is the process of creating genetically identical plants from plant tissue other than male and female gametes. In one approach to somatic cloning, plant tissue is cultured in an initiation medium that includes hormones, such as auxins and/or cytokinins, to initiate formation of embryogenic tissue, such as an embryogenic suspensor mass, that is capable of developing into somatic embryos. An embryogenic suspensor mass, or ESM, has the appearance of a whitish translucent mucilaginous mass and contains a plurality of early stage embryogenic tissue. The embryogenic tissue is further cultured in a multiplication medium that promotes multiplication and mass production of the embryogenic tissue. The embryogenic tissue is then cultured in a development medium that promotes development and maturation of cotyledonary somatic embryos that can, for example, subsequently be placed on germination medium to produce germinants, which in turn can be transferred to soil for further growth. Alternatively, the cotyledonary somatic embryos can be placed within manufactured seeds and sown in soil where they germinate to yield seedlings. Manufactured seeds are described, for example, in U.S. Pat. Nos. 5,564,224; 5,687,504; 5,701,699; and 6,119,395.
The typical somatic embryogenesis process is laborious and inefficient. For example, one of the more labor intensive and subjective steps in the embryogenesis process is the selective harvesting of individual embryos suitable for germination from development medium. At the end of the development period, the embryos may be present in a number of stages of maturity and development. Those that are most likely to successfully germinate into normal plants are preferentially selected using a number of visually evaluated screening criteria. Typically, a skilled technician evaluates the morphological features of each embryo, such as the embryo's size, shape (e.g., axial symmetry), cotyledon development, surface texture, color, and the like, and manually plucks desirable embryos with a pair of tweezers and transfers the selected embryos to germination medium. This is a highly skilled yet tedious job that is time consuming and expensive. Further, it poses a major production bottleneck when the ultimate desired output can be in the thousands of plants.
Automated methods for the harvesting of plant cotyledonary embryos from development medium have been developed, for example, as described in U.S. Pat. No. 7,530,197. At the end of the development phase, the embryos are typically attached to or imbedded in the embryogenic suspensor mass. It is important for subsequent normal germination to separate the embryos from the suspensor mass and from other embryos to yield individual embryos. This can be accomplished by a separation step in which plant embryos are physically separated from each other and the underlying embryogenic suspensor mass before further processing such as, for example, placement onto germination medium.
Separation can be accomplished by washing embryos off of a development medium using aqueous liquid, and then passing the embryos through a porous material, such as a sieve. During sieving, the embryos may be further sprayed with aqueous liquid to facilitate removal and washing away of any undesirable material, such as undersized embryos, tissues, and residual embryonal suspensor mass, through the holes of the porous material, and to sort the embryos according to size. Sorting according to size can be accomplished by using porous materials with various pore sizes. The mesh opening sizes of the sieve(s) can be selected so as to capture the desired sized embryos. The mesh opening sizes may vary in the range from about 500 microns to about 2400 microns. By adjusting the mesh opening size/shape of the one or more sieves, only those embryos within a desirable size/shape range are selected, resulting in a population comprising mostly of a plurality of individual embryos separated from each other and substantially free of suspensor tissue.
Although automated methods have been developed for removing embryos from development medium, and sorting the embryos according to size, technicians are still relied on to select those embryos having characteristics that improve the probability that the selected embryos will successfully germinate into plants; and then to hand-pluck the embryos from the porous material, and transfer the embryos to germination medium. The selection process is highly subjective, and the transferring of embryos to germination medium by hand remains a tedious, laborious, and ergonomically challenging process.
Efforts have been made to use instrumental image analysis for embryo selection to supplement or replace the visual evaluation performed by technicians. For example, an elaborate and complex classification method is disclosed in U.S. Publication No. 2007/0269096, which describes the classification of plant embryos by the application of classification algorithms to digitized images of plant embryos, and absorption, transmittance, or reflectance spectra of the embryos, to determine which embryos are likely to develop into germinants. Similarly, U.S. Pat. No. 7,610,155 describes using image and spectral data from known quality embryos to develop a classification model, using a classification algorithm, such as logistic regression (LR) analysis, to classify embryos as (i) embryos that likely will not germinate; (ii) embryos that may germinate with extra care; and (iii) embryos that will germinate with minimal care. The classification model is then applied to image and/or spectral data acquired from a plant embryo of unknown quality to determine the likelihood the embryo will develop into a germinant. Although determining the germination potential of embryos by classification modeling is a more objective process than selection of embryos by technicians, such methods involve the use of expensive instrumentation to collect the required images and data on each embryo, as well as extensive studies of embryos of known quality to develop the modeling system.
Thus, there exists a need for methods of transferring embryos en masse to germination medium that simplify the process, eliminate the step of determining germination potential of individual embryos, reduce the risk of contamination of the embryos, reduce labor and technician fatigue, reduce the risk of worker injury, and increase the production rate to achieve commercial scale.
The present disclosure describes methods of transferring plant somatic embryos en masse to germination medium.